Compositions for vaginal treatment

ABSTRACT

The invention provides mannose phosphate and salts thereof for increasing vaginal cell growth, vaginal cell maturation and vaginal moisture, as well as compositions, articles and methods for treating and preventing vaginal conditions characterized by poor vaginal cell growth, low vaginal cell differentiation and low vaginal moisture.

RELATED APPLICATION

This application is a divisional under 37° C. F. R. 1.53(b) of U.S.patent application Ser. No. 10/801,063 filed Mar. 15, 2004, whichapplication is incorporated herein by reference and made a part hereof.

FIELD OF THE INVENTION

The invention relates to the use of compositions containing mannosephosphate to promote vaginal cell proliferation and maturation.

BACKGROUND OF THE INVENTION

Vaginal atrophy is a common and well-recognized problem in menopausalwomen. It is found in up to 50% post-menopausal women as well as in10-20% pre-menopausal women with low estrogen levels. Vaginal atrophy isbelieved to be caused by low estrogen levels that result in a decreasein vaginal cell proliferation and differentiation. The decrease in cellproliferation leads to thinning of vaginal epithelium and to a lack ofglycogen production by intermediate cells. Glycogen plays an importantrole in maintaining vaginal ecosystem by serving as food forLactobacilli acidophilus, the normal flora in the vagina, and by servingas a substrate for acid production to maintain low vaginal pH. Thethinning of vaginal epithelium and lack of glycogen in vaginal atrophypatients frequently results in vaginal dryness, discomfort andvaginitis.

While hormone replacement therapy has been used with some success totreat vaginal atrophy, new findings of the Women's Health InitiativeStudy indicate that such hormone replacement therapy increases the riskof heart attacks, stroke, blood clots, and breast cancer.

Therefore, a need exists for non-toxic compositions and methods to treatvaginal atrophy without the need for hormonal replacement therapies thatcan have negative side effects.

SUMMARY OF THE INVENTION

The present invention is directed to compositions and methods fortreating and preventing vaginal conditions characterized by low vaginalcell proliferation, low vaginal cell differentiation, low vaginalmoisture or vaginal atrophy. The compositions and methods of theinvention employ mannose phosphate, which can promote vaginal cellproliferation and maturation.

Therefore, in some embodiments, the invention is directed to acomposition comprising an effective amount of mannose phosphate and apharmaceutically acceptable carrier. The compositions of the inventioncan promote vaginal cell growth, vaginal cell maturation and may be usedto increase the thickness and restore the health of vaginal epithelium.

The invention is also directed to a method for treating or preventing avaginal condition in a mammal. The method involves administering to amammal an effective amount of a composition that includes a mannosephosphate or a salt thereof. As illustrated herein, the mannosephosphate or salt thereof can increase growth of mammalian epithelialcells and promote vaginal cell maturation. The compositions of theinvention can therefore be used to treat conditions such as low vaginalcell proliferation, low vaginal cell differentiation or low vaginalmoisture. In one embodiment, the condition is vaginal atrophy.

In one embodiment, the mannose phosphate employed ismannose-6-phosphate. As is known to one of skill in the art,mannose-6-phosphate can assume a number of conformations, all of whichare contemplated by the invention. Some of the conformations thatmannose-6-phosphate can assume are depicted below:

wherein X is a double bond, hydrogen or a cation. The cation can be amonovalent or divalent cation, for example, sodium, potassium, calcium,magnesium, manganese, zinc and the like.

Moreover, in some compositions of the invention some of the mannosephosphate molecules can have a variety of substituents in place of thehydroxy (—OH), aldehyde

(—CHO), and hydrogen substituents that are generally found in mannosephosphate preparations. In general, the exact type of substituents onthe mannose phosphate compound(s) of the invention can be varied tostimulate optimal levels of vaginal cell growth and/or maturation.Polymers of mannose-6-phosphate that can breakdown into monomers ofmannose phosphate are also contemplated. Such polymers can include othersaccharides or non-saccharides that breakdown to permit release ofmannose phosphate.

The compositions of the invention are generally administeredintravaginally. However, other routes are also contemplated. Forexample, the compositions can also be administered topically. Thecompositions can be incorporated into feminine products such as douches,tampons, foams, creams, sustained release implants and the like for easyuse and administration. The invention further provides syringe-likeapplicators for administration of the compositions of the invention.Such applicators can contain a mannose phosphate composition of theinvention. Douches, tampons, foams, creams, sustained release implantsand applicators can be prepared in a sterile manner to optimize theshelf life of the composition and to permit the composition to bedispensed in a sterile manner.

An effective amount of the compounds of the invention can vary, but insome embodiments the effective amount of mannose phosphate can rangefrom about 0.01 micrograms to about 500 milligrams.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the effect of mannos-6-phosphate (M6P) on cellproliferation in normal human vaginal epithelial cells in culturemedium. Six samples were tested in each group; the error bar representsthe standard deviation.

FIG. 2 illustrates that mannos-6-phosphate (M6P) treatment increases theobserved numbers of mature vaginal epithelial cells as measured byglycogen production. Glycogen production in vaginal epithelial cellcytoplasm is a differentiation/maturation marker for vaginal epithelialcells. Each group had 5 samples and the error bar represents thestandard deviation.

FIG. 3 is a schematic diagram of one type of syringe-like applicatorthat can be used to deliver a mannose phosphate composition to a vaginaof a mammal. The syringe-like applicator consists of a barrel 20 and aplunger 30 with a plunger head 40. The syringe-like applicator can alsohave a barrier seal 60 distal to the plunger head 40. The syringe-likeapplicator can also have a chamber 50 within the barrel that liesbetween the plunger 30 and the barrier seal 60. The chamber 50 comprisesan effective amount of a mannose phosphate compound or a salt thereof.The presence of the barrier seal 60 seals the applicator and keeps thecomposition contained with the applicator during shipping and handling.The barrier seal 60 can be removed by the user, or it can rupture whenthe user depresses the plunger. At the time of use, the applicator isinserted into the vagina and the plunger 30 is depressed. This forcewill push the composition out of the applicator and into the vagina.

FIG. 4 is a schematic diagram of a vaginal insert that can be used todeliver a mannose phosphate composition to the vagina of a mammal. Thevaginal consists of a tubular shaped material 110 comprising a mannosephosphate compound and a string 120 or other attachment for retrieval orpositioning of the vaginal insert within the vagina. In someembodiments, the string 120 is optional, for example, because thevaginal insert slowly dissolves, breaks down or erodes to providesustained release of mannose phosphate. Hence, retrieval may not beneeded. The syringe-like applicator illustrated in FIG. 3 may be usedfor delivery of the vaginal insert.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions and methods to increase vaginal cellproliferation and vaginal cell differentiation or maturation. Suchcompositions can promote vaginal cell growth, the development ofdifferentiated vaginal cells and may be used to increase the thicknessand restore the health of vaginal epithelium. The compositions employedby the invention are inexpensive, non-toxic and readily available. Theefficacy of these compositions and methods does not rely upon hormonalreplacement or substances that are not naturally found in the body.Hence, the compositions avoid the negative side effects associated withcommonly used hormone replacement therapies.

Mannose-Phosphate Compositions

Compositions employed by the invention for increasing vaginal cellproliferation and maturation contain a mannose phosphate, preferablymannose-6-phosphate (M6P).

In some embodiments, other saccharides can also be included in thecompositions of the invention. For example, the mannose-phosphatecompositions can include glucose-1-phosphate, glucose-6-phosphate,mannose-1-phosphate, galactose-6-phosphate, fiuctose-6-phosphate,glucose-1,6-diphosphate, or fructose-1,6-diphosphate. The percentage ofalternate saccharides in the compositions of the invention can vary. Forexample, the compositions of the invention can include 0% to about 50%alternate saccharides. In other embodiments, the compositions of theinvention can include about 0% to about 40%, or 0% to about 30%, or 0%to about 20% or 0% to about 10% alternate saccharides.

Moreover, the mannose phosphate for use in the present invention can beformulated as pharmaceutically or cosmetically acceptable salts thereof,e.g., mono- or disodium salts, as well as any precursor forms that whenapplied to the epithelium or skin release the mannose phosphate.

In some embodiments some of the mannose phosphate sugar units can have avariety of substituents in place of the hydroxy (—OH), aldehyde (—CHO),and hydrogen substituents that are typically found on mannose phosphate.For example, lower alkyl moieties can replace any of the hydrogen atomsfrom the hydroxy (—OH) or hydrogen substituents of the mannose phosphatecompounds employed in the invention. Amino or lower alkyl amino groupscan replace any of the OH or hydrogen groups of the mannose phosphatecompounds employed in the invention. Sulfate (SO₄ ⁻) may replace thephosphate groups of the mannose phosphate compounds employed in theinvention. Hence, substituents that can be present instead of, or inaddition to, the substituents typically present on mannose phosphatecompounds include sulfate (SO₄ ⁻), lower alkoxy, lower alkanoyloxy,and/or lower alkanoylaminoalkyl.

As used herein, lower alkyl means (C₁-C₆) alkyl. Such (C₁-C₆) alkyl canbe methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl,pentyl, 3-pentyl, or hexyl. Preferred lower alkyl groups are (C₁-C₃)alkyl including methyl ethyl, propyl, isopropyl and the like. Loweralkoxy generally means (C₁-C₆) alkoxy; such (C₁-C₆) alkoxy can, forexample, be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy,sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy. Lower hydroxy alkyl refersto a hydroxy group attached to a lower alkyl or lower alkylene group(e.g. —CH₂—CH₂—OH). Lower alkanoyloxy refers to (C₂-C₆) alkanoyloxy, forexample, acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy,pentanoyloxy, or hexanoyloxy. Lower (C₁-C₆) alkanoylamino can, forexample, be acetamino, propanoylamino, butanoylamino, isobutanoylamino,pentanoylamino, or hexanoylamino.

Therefore, while the active ingredients in the compositions of theinvention typically include a high percentage of mannose phosphate, somevariability in the types of substituents and sugar units present in themannose phosphate preparation employed is acceptable so long as thepreparation can increase vaginal cell proliferation or maturation.

In other embodiments, polymers and mixtures of mannose phosphate withother polymers can be employed in the compositions and methods of theinvention. Polymers of mannose phosphate that can breakdown to releasemannose phosphate monomers are particularly desirable. In someembodiments, non-saccharide polymers can be used in the mannosephosphate formulations. Examples of polymers that can be used withmannose phosphate include proteoglycans, poly(ethyleneglycol)/poly(ethylene oxide), poly(vinyl alcohol),poly(vinylpyrrolidone), and poly(2-hydroxylethyl methacrylate). Suchpolymers can be covalently bonded to mannose phosphate or simplycombined with mannose phosphate to form a mixed composition.

Methods of Use

The present invention is directed to methods of treating or preventingor otherwise ameliorating vaginal conditions characterized by poorvaginal cell growth, low vaginal moisture and poor vaginal celldifferentiation. The compositions of the invention can also be used invaginal moisturizers and in methods for moisturizing the vagina torelieve vaginal dryness and restore health to the vaginal epithelium.

In some embodiments, the compositions and methods of the invention areused in subjects with an abnormally thin vaginal lining or subjects withan abnormally thin vaginal mucosa. Symptoms resulting from theabnormally thin vaginal lining or mucosa include vaginal dryness,discomfort, itching, dyspareunia, infection, inflammation, ulcers,discharge, and bleeding. In some instances, the compositions and methodsof the invention are used to treat vaginal atrophy.

Vaginal atrophy is a condition occurring in some women, typicallypostmenopausal women, in which there is significant thinning of themucosa of the vagina. The thin vaginal mucosa lacks maturation, meaningthat it consists of numerous parabasal cells and little or nosuperficial and intermediate cells, which results in decreased glycogendeposits and a higher pH. Vaginal atrophy is caused chiefly by anestrogen deficiency because the mucosa of the vagina is an estrogensensitive tissue and a well-known target organ for estrogen. As providedherein, vaginal atrophy and the effects of low estrogen on the vaginacan be reversed by treatment with the compositions and methods of theinvention.

These methods include administering to the animal an effective amount ofmannose phosphate, for example, a therapeutically effective amount ofmannose phosphate. According to the invention, a therapeuticallyeffective amount of mannose phosphate can increase vaginal epithelialcell growth, increase the percentage of differentiated or mature vaginalepithelial cells and increase vaginal moisture.

Treatment of, or treating, a vaginal condition is intended to includethe alleviation of or diminishment of at least one symptom typicallyassociated with the vaginal condition, for example, at least one symptomtypically associated with vaginal atrophy. The treatment also includesalleviation or diminishment of more than one symptom. Ideally, thetreatment cures, e.g., substantially eliminates one or more symptomsassociated with the vaginal condition.

Administration

According to the invention, mannose phosphate compounds, polymers,co-polymers, polymer mixtures and their salts, can promote vaginal cellproliferation and/or vaginal cell maturation in animals. The term“animal,” as used herein, refers generally to a warm-blooded animal.Mammals include cattle, buffalo, sheep, goats, pigs, horses, dogs, cats,rats, mice, rabbits, chickens, turkeys, and humans. Also included areother livestock, domesticated animals and captive animals.

An effective amount of a mannose phosphate compound, polymer,co-polymer, polymer mixture or salts thereof, for promoting vaginal cellgrowth and/or maturation is an amount that increases the growth of apopulation of vaginal cells, for example, epithelial cells, relative toa population of the same type of vaginal cells that received no mannosephosphate. To achieve the desired inhibition, the composition may beadministered as single or divided dosages, for example, of at leastabout 0.001 μg to about 100 to 200 mg, of about 0.01 μg to about 75 to100 mg, of about 0.1 μg to about 50 to 75 mg or about 1.0 μg to about 30to about 50 mg of one or more mannose-6-phosphate compound, althoughother dosages may provide beneficial results. In some embodiments, thedosage can vary from about 0.01 mg to about 50 mg.

Daily doses of the compositions of the invention can vary as well. Suchdaily doses can range, for example, from about 0.001 mg/day to about 500mg/day, from about 0.01 mg/day to about 250 mg/day, from about 0.01mg/day to about 120 mg/day, from about 0.1 mg/day to about 100 mg/day,from about 0.1 mg/day to about 75 mg/day, and from about 0.1 mg/day toabout 50 mg/day of one or more of the mannose phosphate compounds.

The amount administered will vary depending on various factorsincluding, but not limited to, the disease, the weight, the physicalcondition, the health, the age of the animal, and whether prevention ortreatment of vaginal atrophy is to be achieved. Such factors can bereadily determined by the clinician employing animal models or othertest systems that are available in the art.

The compositions of the invention can be administered for prophylactic,therapeutic, and/or hygienic use. Such administration can be topical,vaginal, rectal, and other convenient routes. Administration can bedirectly to epithelial cell surfaces. For example, the compositions ofthe invention can be administered directly to mucosal surfaces. Mucosalsurfaces include vaginal, urogenital, rectal and the like. Surfaces ofthe urogenital tract that can be treated with the compositions andmethods of the invention include rectal, urethral, ureteral, vaginal,cervical, uterine, etc. In many embodiments, the epithelial cell surfaceis vaginal.

Administration of the therapeutic agents in accordance with the presentinvention may be in a single dose, in multiple doses, in a continuous orintermittent manner, depending, for example, upon the recipient'sphysiological condition, whether the purpose of the administration istherapeutic or prophylactic, and other factors known to skilledpractitioners. For prevention of certain conditions or diseases (e.g.vaginal atrophy), administration of the compositions of the inventionmay be essentially continuous over an indeterminate period of time, forexample, at regular intervals for life. Alternatively, the compositionsof the invention can be administered continuously for a pre-selectedperiod of time or in a series of spaced doses. Local administration isgenerally contemplated.

The compositions are prepared by combining the active ingredients in theappropriate concentrations. Other active or inactive agents selected byone of skill in the art can optionally be added. The absolute weight ofa given active agent included in a unit dose can vary widely.

The compositions of the invention can be administered in the form of anarticle or carrier such as a vaginal insert, or by way of a syringe-likeapplicator, tablet, suppository, pessary, powder/talc or other solid,solution, liquid, spray, aerosol, douche, ointment, tampon, foam, cream,gel, paste, microcapsule(s), vaginal sponge, vaginal ring, controlledrelease formulation, sustained release formulation or bioadhesive gel(e.g., a mucoadhesive thermogelling composition (see, for example, U.S.application Ser. No. 10/135,805, filed on Apr. 30, 2002, which isincorporated herein by reference)).

For intravaginal administration, the therapeutic agents may beformulated as is known in the art for direct application to the vaginalarea. Forms chiefly conditioned for vaginal application take the form,for example, of creams, milks, gels, dispersions, micro-emulsions,lotions thickened to a greater or lesser extent, impregnated pads,ointments, aerosol formulations (e.g., sprays or foams), creams,lotions, pastes, jellies, sprays, and aerosols. Alternatively, thecomposition can be formulated to be part of an adhesive polymer, such aspolyacrylate or acrylate/vinyl acetate copolymer.

Ointments and creams may, for example, be formulated with an aqueous oroily base with the addition of suitable thickening and/or gellingagents. Lotions may be formulated with an aqueous or oily base and willin general also contain one or more emulsifying agents, stabilizingagents, dispersing agents, suspending agents, thickening agents, orcoloring agents. Liquid sprays are conveniently delivered frompressurized packs, for example, via a specially shaped closure. Theactive compositions can also be delivered via iontophoresis, e.g., asdisclosed in U.S. Pat. Nos. 4,140,122; 4,383,529; or 4,051,842. Thepercent by weight of a prophylactic agent of the invention present in aformulation will depend on various factors, but generally will be fromabout 0.01% to about 98% of the total weight of the formulation, andtypically about 0.1 to about 90% by weight.

The pharmaceutical formulations of the present invention may include, asoptional ingredients, pharmaceutically acceptable carriers, diluents,solubilizing or emulsifying agents, and salts of the type that areavailable in the art. Examples of such substances include normal salinesolutions such as physiologically buffered saline solutions and water.Specific non-limiting examples of the carriers and/or diluents that areuseful in the pharmaceutical formulations of the present inventioninclude water and physiologically acceptable buffered saline solutionssuch as phosphate buffered saline solutions with a pH of about 4.5 toabout 5.5.

Furthermore, the active ingredients may also be used in combination withother therapeutic agents, for example, anti-microbial agents, painrelievers, anti-inflammatory agents, vitamins (e.g., vitamin B, C or E),aloe vera and the like, whether for conditions described herein or someother condition. Previous work by the inventors has shown thathyaluronic acid compositions can inhibit pathogen attachment to avariety of cell types, including vaginal epithelial cells. See U.S. Ser.No. 10/401,522 and U.S. Ser. No. 10/608,848, which are incorporatedherein by reference. Moreover, the inventors have shown that hyaluronicacid can promote vaginal cell growth and maturation. See U.S. Ser. No.10/696,547, which is incorporated herein by reference. Hence, accordingto the invention, compositions containing mannose phosphate can alsocontain hyaluronic acid.

The present invention further pertains to a packaged pharmaceuticalcomposition for controlling or preventing a vaginal condition (e.g.vaginal atrophy) such as a kit or other container. The kit or containerholds a therapeutically effective amount of a pharmaceutical compositionfor preventing, controlling or inhibiting the vaginal condition andinstructions for using the pharmaceutical composition for prevention,control or inhibition of the vaginal condition. The pharmaceuticalcomposition includes mannose phosphate, in a therapeutically effectiveamount such that the vaginal condition is prevented, controlled orinhibited.

In addition, the invention provides a vaginal insert that can releasethe mannose phosphate in a controlled fashion. Such a vaginal insert canbe biodegradable or non-biodegradable. The vaginal insert providessustained release of the active ingredients at an appropriate rate forachieving the desired degree of vaginal cell growth or vaginal cellmaturation, or of the desired degree of vaginal atrophy prevention ortreatment.

In some embodiments, the active ingredients can be formulated witholeaginous bases or ointments to form a semisolid composition with adesired shape. For example, the composition can be shaped for easyinsertion into an orifice such as the vagina. This class of formulationscomprises the active ingredients and hydrocarbon-based semisolidscontaining dissolved and/or suspended bacteriostats/preservatives and abuffer system. The petrolatum component in these bases can be anyparaffin ranging in viscosity from mineral oil employing incorporatedisobutylene, colloidal silica, or stearate salts to paraffin waxes.White and yellow petrolatums are examples of such petrolatum components.Bases of this class can be made by incorporating high-melting waxes intoa fluid mineral oil via fusion or by incorporation of polyethylene intomineral oil at elevated temperature. Polysiloxanes (also known assilicones) are suitable for use in these bases and typically have aviscosity in the range of about 0.5 to 10⁶ centistokes. The organicentities attached to the polysiloxane are preferably lower molecularweight hydrocarbon moieties having from 1 to 8 carbons each, such aslower alkyl, lower alkenyl, phenyl and alkyl substituted phenyl, andphenyl(lower)alkyl, such as benzyl. In such a moiety, each lower alkylor alkenyl group preferably has 1 to 3 carbons inclusive, such as in adimethylsiloxane polymer.

Absorption bases can be used with such an oleaginous system. In additionto the active ingredients, additional ingredients with the capacity toemulsify a significant quantity of water are employed. Water-in-oil(w/o) emulsions can be formed wherein the external phase is oleaginousin character. Preservatives/bacteriostats, such as the parabens, buffersystems, etc. can be incorporated into these bases as emulsified aqueoussolutions together with the active ingredient. Diverse additives areconveniently used as the emulsifier, and these include, but are notlimited to, cholesterol, lanolin (which contains cholesterol andcholesterol esters and other emulsifiers), lanolin derivatives, beeswax,fatty alcohols, wool wax alcohols, low HLB (hydrophobe/lipophobebalance) emulsifiers, and assorted ionic and nonionic surfactants,singularly or in combination.

Water-In-Oil emulsion bases can be employed in the compositions, insertsand articles of the invention. These formulations can be an expansion ofthe general class of absorption bases that includes liquids or creams.They can be prepared by taking a mixture of the active ingredients withoil phase ingredients, bacteriostats/preservatives and buffer salts thatare dissolved or suspended therein and to which water has been added toform a water-in-oil emulsion.

Oil-In-Water emulsion bases can also be utilized in the compositions,inserts and articles of the invention. These systems are semisolidemulsions, micro-emulsions, or foam emulsion systems. Usually such asystem has a “creamy white” appearance. Typically, the internal oilphase is in the range in percentage composition of about 10% to about40% oil by weight and the external phase may contain 80% or more water.The oleaginous phase may contain, but is not limited to, long-chainalcohols (cetyl, stearyl), long-chain esters (myristates, palmitates,stearates), long-chain acids (palmitic, stearic), vegetable and animaloils and assorted waxes. These can be made with anionic, cationic,nonionic or amphoteric surfactants, or with combinations especially ofthe nonionic surfactants.

Inserts and suppositories containing the active ingredients can be, forexample, oleaginous in nature that melt at body temperature, orpolyethylene glycol-based compositions that dissolve in mucosal (e.g.vaginal) fluids. Additional bases for suppositories are glycerin andglycerinated gelatin.

The active ingredients can be formulated into inserts, articles,tampons, transdermal patches, bandages, and dressings using bufferedgels made with gelling agents. Some examples of these gelling agentsare: cellulosics, cationic polymers, polyoxyalkylenes, and carboxyvinylpolymers. Cellulosics useful in the formulations of the inventioninclude, for example, methyl cellulose, carboxymethyl cellulose,hydroxyethyl cellulose, and hydroxypropyl cellulose. Cationic Polymersuseful in the formulations of the invention include “Polyquaternium-10”,a polymeric quaternary ammonium salt of hydroxyethyl cellulose reactedwith a trimethyl ammonium-substituted epoxide, and the like.Polyoxyalkylenes useful in the invention includepolyoxyethylene-polyoxypropylene esters of lanolin and derivativesthereof. Carboxyvinyl polymers useful for the formulations of theinvention include cross-linked acrylic acid polymers, e.g., thosecommercially available from B. F. Goodrich Co., Akron, Ohio, under thedesignation CARBOPOL™.

Controlled or sustained release can be achieved by the addition oftime-release additives, such as polymeric structures, matrices, that areavailable in the art. For example, the compositions of the invention mayalso be administered through the use of hot-melt extrusion articles,such as bioadhesive hot-melt extruded film (see, for example, U.S. Pat.No. 6,375,963, which is incorporated herein by reference). Theformulation can comprise a cross-linked polycarboxylic acid polymerformulation, generally described in U.S. Pat. No. 4,615,697 to Robinson(hereinafter “the '697 patent”), which is incorporated herein byreference. In general, about eighty percent of the monomers of thepolymer in such a formulation contain at least one carboxylfunctionality. The cross-linling agent should be present at such anamount as to provide enough adhesion to allow the system to remainattached to the target epithelial or endothelial cell surfaces for asufficient time to allow the desired release of mannose phosphate totake place.

An insert or article can comprise a mixture or coating of polymers thatprovide release of the active agents at a constant rate over a prolongedperiod of time. In some embodiments, the article or insert compriseswater-soluble pore forming agents, such as polyethylene glycol (PEG)that can be mixed with water insoluble polymers to increase thedurability of the insert and to prolong the release of the activeingredients. Such a water-soluble pore-forming agent can be polyethyleneglycol, polypropylene glycol, a mixture or polymer of sugars (lactose,sucrose, dextrose, etc.), salts, poloxamers, hydroxypropylcellulose,polyvinyl alcohol and other water-soluble food grade and otherexcipients.

When PEG is used as a pore forming agent, the molecular weight of PEG isin the range from about 200 to about 20,000, alternatively, from about400 to about 8,000. For example, PEG having a molecular weight of about540 to about 8,000 is used. In another embodiment, the PEG has amolecular weight of about or above 1,000 to about 8,000. The molecularweight of PEG used for the coating with the formulation of the inventionwill depend on the ability of PEG to form a coating film that isnon-sticky, having enough strength and creating adequate pore size forcontrolling the release of active ingredients over the desired timeperiod both in vitro and in vivo.

The pore-forming agent is used in the formulation of the invention inthe amount effective to regulate the release of a mannose phosphatecompound at a desired rate. Preferably, the effective amount of thepore-forming agent provides long term delivery of the active agent thusincreasing the useful life of a sustained-release insert, article orimplant. The effective amount of the pore forming agent will depend onthe desired rate and duration of the release and the ability to form acontinuous microporous film during the coating process. To enablerelease duration over longer periods of time PEG with higher molecularweights is used. For example, PEG 8000 can provide release over a periodof time that is longer than 100 days, when used in a concentration from10 to 50%, preferably from 20 to 45% and most preferably from 30 to 45%.The concentration of PEG is expressed herein in % weight per dry basisand represents the concentration of PEG in the coating film afterdrying. Similarly, the thickness of the coating film is from 5 to 50 μm,preferably 30 from 10 to 30 μm and most preferably from 15 to 25 μm.

A good correlation exists between the dissolution rate of active agentsand the amount of pore forming agent incorporated in the coating filmbased on in vitro and in vivo studies. Depending on the desired lengthof release, the PEG concentration ranges can be adjusted as needed. Forexample, in vivo duration of a coated insert may be predicted simplyfrom the in vitro dissolution rate of the active agent at the 120-hourtime point.

The inserts and articles of the invention may also comprise a waterinsoluble polymer. Examples of such polymers are ethylcellulose, acrylicresins, co-polymer of methacrylic acid and acrylic acid ethyl ester,polylactic acid, PLGA, polyurethane, polyethylene vinyl acetatecopolymer, polystyrene-butadiene copolymer and silicone rubber, ormixtures thereof. For example, polymers sold under trade names AquacoatECD 30 and Eudragit RS 30 and NE 30D (registered trademarks of RhomTech, Inc.) can be used.

A polymer suitable for use in this invention is a polymer that canrelease the active agents of the invention at a rate that is sufficientfor treatment of a vaginal condition described herein. The ratecontrolling formulation prepared with such a polymer is stable duringimplantation. The formulation should have enough strength to withstandroutine handling, and have the stability to release the activeingredients at an acceptable rate.

In one embodiment, the coating formulation of the invention is used tocoat pellets comprising the active ingredients that are compressed toform a solid, biodegradable insert and then administered for promotingvaginal cell proliferation or maturation.

A polymer formulation can be utilized to provide controlled or sustainedrelease. Such a polymer formulation can be adjusted to control therelease rate of the mannose phosphate by varying the amount ofcross-linking agent in the polymer. Suitable cross-linking agentsinclude divinyl glycol, divinyibenzene, N,N-diallylacrylamide,3,4-dihydroxy-1,5-hexadiene, 2,5-dimethyl-1,5-hexadiene and similaragents.

One example of a polymer for use in such a formulation is Polycarbophil,U.S.P., which is commercially available from B. F. Goodrich SpecialtyPolymers of Cleveland, Ohio under the trade name NOVEON™-AA1. The UnitedStates Pharmacopeia, 1995 edition, United States PharmacopeialConvention, Inc., Rockville, Md., at pages 1240-41, indicates thatpolycarbophil is a polyacrylic acid, cross-linked with divinyl glycol.

Other useful bioadhesive polymers that may be used in such a drugdelivery system formulation are mentioned in U.S. Pat. No. 4,615,697.For example, these include polyacrylic acid polymers cross-linked with,for example, 3,4-dihydroxy-1,5-hexadiene, and polymethacrylic acidpolymers cross-linked with, for example, divinyl benzene. Typically,these polymers would not be used in their salt form, because this woulddecrease their bioadhesive capability. Such bioadhesive polymers may beprepared by conventional free radical polymerization techniquesutilizing initiators such as benzoyl peroxide, azobisisobutyronitrile,and the like. Exemplary preparations of useful bioadhesives are providedin the '697 patent.

For vaginal administration, the formulation preferably degrades slowlyor remains attached to the epithelial or endothelial cell surfaces for aperiod of at least about one to about forty-eight hours. Such resultsmay be measured clinically over various periods of time, by testingsamples from the vagina for mannose phosphate. Bioadhesion of aformulation of the invention can be attained with bioadhesive polymersusing a cross-linking agent that is present at about 0.1 to 6.0 weightpercent of the polymer, with about 1.0 to 2.0 weight percent in someembodiments, to achieve an appropriate level of bioadhesion. Bioadhesioncan also be measured by commercially available surface tensiometersutilized to measure adhesive strength.

The formulation may be in the form of a gel, cream, tablet, capsule,suppository, film, or any other pharmaceutically acceptable form that istolerated by epithelial cells (e.g. the mucosa) and does not wash awayeasily. Different formulations are further described in U.S. Pat. No.4,615,697, which is incorporated herein by reference.

As will be apparent to those skilled in the art, the composition of theformulation can be varied to affect certain properties of theformulation. For example, the viscosity can be varied by adding apolymer or gel former. In some embodiments, a bioadhesive polymer can beincluded at various concentrations to provide greater or lesserbioadhesion. A pH sensitive bioadhesive can be utilized to effectgreater release at certain pH values. The particular bioadhesivequalities should prevent the composition from being diluted or washedaway, thereby increasing the utility of the present formulation.

Liquid compositions of the invention can be administered from absorbentmaterials, such as a bandage, tampon or sponge, or as a spray/aerosol(applied to the affected area using a pump-type or aerosol sprayer). Theuse of a tampon, in which the composition of the invention has beenincorporated, is advantageous in that it the composition will be slowlyand continuously released even though it may be continuously carriedaway by menstrual blood or other vaginal discharge. Providing thecomposition in the form of a solution, which may initially be providedin a concentrated liquid form, or as a dissolvable powder, tablet or thelike requiring the addition of water, saline or other suitable diluentsprior to use, enables the composition to be administered as a vaginaldouche.

Solid compositions of the invention can be applied by any number ofmeans, including the use of applicators or by patient self-insertion.For example, creams, lotions, suppositories, foams, pastes, ointments,gels, tablets, or tampons may be administered using an applicator, suchas a squeeze-type or plunger-type applicator. Administering thecomposition as a vaginal suppository is advantageous as it providesconvenience, ease of application, increased safety and/or neatness.Administering the composition as a cream having low surface tension isadvantageous as it provides a uniform wetting action that assists incomposition penetration into crypts and crevices of the orifice. Such acreamy composition can also act as a moisturizer.

Additionally, additives may be mixed in with the formulation for maximumor desired efficacy of the delivery system or for the comfort of thepatient. Such additives include, for example, lubricants, plasticizingagents, preservatives, gel formers, tablet formers, pill formers,suppository formers, film formers, cream formers, disintegrating agents,coatings, binders, vehicles, coloring agents, taste and/or odorcontrolling agents, humectants, viscosity controlling agents,pH-adjusting agents, and similar agents.

One desirable embodiment provides for compositions of the invention in asyringe-like applicator (also known as a plunger-type or syringe-likeapplicator (see FIG. 3)). For example, a composition including mannosephosphate may be placed into a chamber 50 within the barrel 20 of asyringe-like applicator. The chamber is sealed at the distal end with abarrier seal 60 and at the proximal end by the plunger head 40. Thepresence of the barrier seal 60 seals the applicator and keeps thecomposition contained with the applicator during shipping and handling.However, the barrier seal 60 can be removed by the user or it canrupture when the user depresses the plunger 30. At the time of use, theapplicator is inserted into the vagina and the plunger 30 is depressed.This force will push the composition out of the applicator and into thevagina. As an alternative, a tapered tip can be used in place of thebarrier seal 60.

One embodiment of the invention provides an aqueous gel containing amucoadhesive material, such as carboxymethylcellulose (optionally mixedwith a thermogelling mucoadhesive agent), to be mixed with mannosephosphate to thereby form a composition of the invention. An additionalembodiment provides for the encapsulation of mannose phosphate inpolymeric microparticles. Once in situ, the polymer dissolves and themannose phosphate is released. In this case, release of mannosephosphate can be controlled by the microparticles to provide extendedproduction of the desired product (e.g., sustained release). Thedelivery vehicle is not limited to use in the vagina, but could also beapplied to a wide variety of biomedical applications where delivery ofmannose phosphate is desired. Appropriate modification of the deliveryvehicles described herein is within the skill of those in the art.

Additionally, the composition and/or delivery materials may containadditional beneficial agents that can improve the health of the vagina.For example, polymers used as carrier or for encapsulation or forsustained release may be hydrolytically degraded into an acid or acidproducing species. One such polymer is a poly(vinyl alcohol) backbonewith pendant polycaprolactone chains that, upon disintegration, yieldspoly [vinyl(polyeaprolactate)]. The polycaprolactone is hydrolyticallydegraded into caproic acid. This acid aids in lowering pH andcontrolling harmful bacterial growth, thus helping to restore balance tothe epithelium. In addition, this material is melt processable and canbe formed into a system for controlled delivery of the mannosephosphate. Additionally, a peroxide of Laureth-4 (e.g., a Laureth-4terminal peroxide) would release laureth-4 and peroxide (e.g., hydrogenperoxide). Laureth-4 decreases TSS-1 production by S. aureus and theperoxide is available to suppress undesirable anaerobes and Gardnerellavaginalis, thus reducing toxin production while reestablishing thevaginal flora.

The Examples further illustrate certain aspects of the invention and arenot intended to limit the invention in any manner.

EXAMPLE Mannose-6-Phosphate Promotes Vaginal Cell Proliferation andMaturation

This Example describes experiments showing that mannose phosphates canpromote vaginal cell proliferation and vaginal cell maturation.

Materials and Methods

Normal human vaginal epithelial cells from Clonetics (NHVE 5164) weresubcultured in basal PrEBM (Clonetics CC 3165) within 96 well plates at37° C., 5% CO₂. Cells were then exposed to medium containingmannose-6-phosphate (Sigma, M3655) at a concentration of 10⁻⁵ M. Controlgroups received culture medium without mannose-6-phosphate. Cellproliferation was examined three days later and glycogen positive cellswere detected five days after mannose-6-phosphate treatment.

Cell proliferation was examined by using CellTiter 96 Aqueous OneSolution from Promega (#TB245). Twenty μl of the reagent was added toeach well. The plates were returned to cell culture incubator for 3hours. The absorbance in each well was measured at 490 nm with amicrotiter reader.

A periodic acid Schiff (PAS) assay was used to detect glycogen levels.Before the assay, cells were washed with PBS and fixed with a 10 vol %Formalin/ethanol solution for 1 hour at room temperature, followed bycontinuous rinsing with deionized (DI) water for 1 minute. A Harleco®PAS Assay Kit (from EM Science 64945/43) was used for glycogendetection. The basic principle is that the hydroxy groups on theglycogen units were first oxidized into aldehyde groups by periodicacid, and the aldehyde groups reacted with Schiff's agent to produce ared colorant. The specific assay operations were all done at roomtemperature as follows:

-   -   1. Periodic acid reagent was added to the well/chamber and        allowed to stand for 10 minutes.    -   2. Samples were continuously rinsed in water for one minute.    -   3. Schiff's reagent was added and the samples were allowed to        stand for 15 minutes.    -   4. Samples were continuously rinsed in deionized water for one        minute.    -   5. Sodium carbonate reagent (diluted 2× with deionized water)        was added and samples were allowed to stand for 5 minutes.    -   6. Samples were continuously rinsed in deionized water for 5        minutes.    -   7. Light Green SF Yellowish reagent was added and samples were        allowed to stand for 30 seconds.    -   8. Samples were rinsed in deionized water briefly for 10        seconds.    -   9. Samples were dehydrated with a series of ethanol/water        mixtures (75%, 90%, 95% to 100% ethanol) and xylene.    -   10. Samples were mounted with mounting medium.        The numbers of glycogen-stained positive cells were manually        counted under microscope. The data was analyzed by student's t        test, and p<0.05 was considered significant.        Results

As shown in FIG. 1, mannose-6-phosphate treatment significantly (p<0.05)increased vaginal cell proliferation. Moreover, the number ofglycogen-positive cells was significantly higher when vaginal cells weretreated with mannose-6-phosphate (FIG. 2).

These results indicate that mannose-6-phosphate can stimulate cellproliferation and vaginal cell maturation. Vaginal atrophy is thethinning of the vaginal epithelium that may be caused by low estrogenlevels resulting in a decrease in vaginal cell proliferation. Atrophicvaginitis, the overgrowth of pathogens due to the thinning of protectiveepithelium, can evolve in patients having vaginal atrophy. Hence,compositions containing mannose-6-phosphate that stimulate vaginal cellproliferation may be used to increase the thickness of vaginalepithelium, stimulate maturation of differentiated vaginal cells andrestore healthy vaginal epithelium.

All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each such referenced patent or publicationis hereby incorporated by reference to the same extent as if it had beenincorporated by reference in its entirety individually or set forthherein in its entirety. Applicants reserve the right to physicallyincorporate into this specification any and all materials andinformation from any such cited patents or publications.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and that they are not necessarily restricted to theorders of steps indicated herein or in the claims. As used herein and inthe appended claims, the singular forms “a,” “an,” and “the” includeplural reference unless the context clearly dictates otherwise. Thus,for example, a reference to “an antibody” includes a plurality (forexample, a solution of antibodies or a series of antibody preparations)of such antibodies, and so forth. Under no circumstances may the patentbe interpreted to be limited to the specific examples or embodiments ormethods specifically disclosed herein. Under no circumstances may thepatent be interpreted to be limited by any statement made by anyExaminer or any other official or employee of the Patent and TrademarkOffice unless such statement is specifically and without qualificationor reservation expressly adopted in a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

1. A vaginal insert for increasing vaginal cell growth in a mammalcomprising an effective amount of mannose phosphate or a salt thereof,and a carrier, wherein the effective amount is about 0.001 μg to about500 mg of mannose phosphate or a salt thereof.
 2. The vaginal insert ofclaim 1, wherein the insert can increase production of glycogen byvaginal epithelial cells.
 3. The vaginal insert of claim 1, wherein theinsert is used to treat or prevent low vaginal cell proliferation, lowvaginal cell differentiation or low vaginal moisture.
 4. The vaginalinsert of claim 1, wherein the insert is used to treat or preventvaginal atrophy.
 5. The vaginal insert of claim 1, wherein the mannosephosphate is mannose-6-phosphate.
 6. The vaginal insert of claim 1,wherein the mannose phosphate is a compound of the formula:

wherein X is a monovalent or divalent cation.
 7. The vaginal insert ofclaim 1, wherein the mannose phosphate is mixed with or covalentlylinked to a polymer.
 8. The vaginal insert of claim 7, wherein thepolymer is poly(ethylene glycol), poly(vinyl alcohol),poly(vinylpyrrolidone), or poly(2-hydroxylethyl methacrylate).
 9. Thevaginal insert of claim 1, wherein the composition further comprises ananti-bacterial, anti-fungal or anti-viral agent.
 10. The vaginal insertof claim 1, wherein the composition further comprises hyaluronic acid.11-21. (canceled)